STOCHASTIC MODELING OF BIOLOGICAL PROCESSES: QUANTITATIVE OPTICAL IMAGING AND TUMOR-INDUCED ANGIOGENESIS
, Head, Section on Biomedical Stochastic Physics
Victor Chernomordik, PhD, Staff Scientist
Franck Amyot, PhD, Research Fellow
Moinuddin Hassan, PhD, Research Fellow
Jason Riley, PhD, Postdoctoral Fellow
Alex Small, PhD, Postdoctoral Fellow
Alexander Sviridov, PhD, Guest Researcher 1
Abby Vogel, MS, Intramural Research Training Award Student 2
Daniel Ortanez, Summer Student 3
Zachary Ulissi, Summer Student 4
We devise quantitative theories, develop methodologies, and design instrumentation to study biological phenomena that are characterized by elements of randomness in both space and time. In developing quantitative theories applicable to quantitative optical spectroscopy and tomographic imaging of tissues, we analyze different optical sources of contrast such as endogenous or exogenous fluorescent labels, absorption (e.g., hemoglobin or chromophore concentration), and/or scattering. We design and conduct experiments and computer simulations to validate our theoretical findings. In collaboration with other scientists at the NIH and researchers around the country and world, we investigate physiological sites for which optical techniques might be clinically practical while yielding new diagnostic knowledge and/or generating less morbidity than existing diagnostic methods. Given that angiogenesis plays an essential role in establishing tumor malignancy, we try to establish quantitative methods and in vitro assays to study the mechanisms underlying angiogenesis through stochastic modeling.
Quantitative characterization of tissue
Biological tissues often exhibit characteristic regular features or ornamental patterns. Transition from normal tissue function to diseased tissue can be detected by quantifying irregular patterns. The degree of statistical similarities in a region of interest can carry valuable comparative information about the structural features of the tissue and help characterize tissue and analyze disease localization and progression.
To visualize subsurface structural features of biological tissues, we developed a user-friendly polarization imaging system that simultaneously images cross- and copolarized light. We also developed a quantitative statistical tool, based on Pearson’s correlation coefficient analysis, to enhance the image quality and reveal regions of high statistical similarities within noisy tissue images. We showed that, under certain conditions, maps of the correlation coefficient are determined by the textural character of tissues rather than by choice of the reference image region, thereby providing information on tissue structure. We are testing such a polarized imaging system in combination with methods for the visualization of hidden structures of various tissues and plan to use the system for noncontact sensing of vaginal mucosa abnormalities and skin fibrosis in the clinical setting.
Sviridov A, Chernomordik V, Hassan M, Russo A, Eidsath A, Smith P, Gandjbakhche A. Intensity profiles of linearly polarized light backscattered from skin and tissue-like phantoms. J Biomed Opt 2005;10:014012.
Sviridov AP, Chernomordik V, Hassan M, Boccara AC, Russo A, Smith P, Gandjbakhche A. Enhancement of hidden structures of early skin fibrosis using polarization degree patterns and Pearson correlation analysis. J Biomed Opt 2005;10:051706.
Sviridov AP, Ulissi Z, Chernomordik V, Hassan M, Gandjbakhche AH. Visualization of biological texture using correlation coefficient images. J Biomed Opt Lett 2006;11:060504.
Fluorescence lifetime imaging
Fluorophore lifetime imaging is a promising tool for studying the tissue environment associated with tumors. The lifetime (time for an electron to return from an excited state to its initial state) of a fluorophore can vary in response to changes in the immediate environment, such as temperature, pH, tissue oxygen content, nutrient supply, and bioenergetic status. Mapping the lifetime and location of a fluorophore in tissue at different depths can be used to monitor these parameters.
We developed a time-resolved lifetime imaging system for small-animal studies that maps fluorophore lifetimes. It consists of a single-source–multiple-detector array that scans the surface of the tissue. Using several source-detector separations, the system probes different depths of the medium. We used a pH-sensitive dye in the near-infrared region to study the tumor environment below the skin surface and demonstrated that, with simplified back projections, we could map the near-surface fluorescent lifetime in vivo. Combining the resultant map with the pre-calibrated lifetime response to pH, we showed that it is possible to perform biologically plausible, non-invasive quantification of pH in mouse tumors. We plan to continue our studies to validate our method by using other pH-monitoring methods. The system offers potential for localizing a tumor and monitoring its status noninvasively in vivo.
We are collaborating with Jacek Capala, who has developed probes in the near-infrared region for noninvasive in vivo monitoring of HER2-positive cancers (for example, breast cancer) and HER2-specific delivery of therapeutic agents. We injected HER2-positive cancer cell lines into the flank area of female nude mice. The cell lines expressed high levels of the HER2 (HER2/neu, c-ErbB2) protein, a 185 kD transmembrane receptor with tyrosine kinase activity that stimulates cell signaling pathways to increase cell proliferation, mobility, and survival. We are investigating the distribution of the probes in vivo by using optical beacons and are quantifying HER2 receptors on tumor cells to assess the response of tumor cells to target therapies aimed at epidermal growth factor signaling pathways.
The analysis of deeply embedded tissue abnormalities by means of time-resolved fluorescence needs to account for the high-scattering nature of biological tissues. To address such tissue properties, we investigated the limitations of a previously developed analytical model of photon migration for localized fluorophores. We showed that the model should incorporate a more realistic distribution of fluorescence lifetimes in order to provide a better description of experimental results, thus providing more flexibility for the inverse model to converge. We have used such an analytic model as a forward model to reconstruct the lifetime and location of a point-source fluorophore. We are now extending the inverse model so that it can handle distributed sources and hence extend current subsurface environmental maps of tissue behavior to deeper tissue imaging. We have made other advances in studying the noise sensitivity of different data types in time-resolved fluorescence imaging, suggesting a new local set of data types that is likely to provide more stability to noise than classical statistical (global) data types used in diffuse optical tomography. We pursued another approach to quantification of fluorescence lifetimes of deeply embedded fluorophores that is applicable when the intrinsic lifetime of the fluorophore is comparable to the photon migration time in the medium. We found and experimentally verified scaling relations that could be used to correct observed time-resolved intensity distributions from fluorescent targets at a given depth z inside turbid medium to an expected surface distribution (from the same fluorophore), revealing the intrinsic fluorescence lifetime without the need for full-scale reconstruction. Similar corrections are useful for comparing the time-resolved data obtained by several detectors from the same deeply embedded fluorophore when the detectors are positioned at different distances from the source (excitation photon entry point into the medium). The random walk model of time-resolved fluorescence imaging substantiates the scaling relations.
Hassan M, Gannot I, Chernomordik VV, Smith PD, Pursley R, Gandjbakhche AH. A scanning system for fluorescence lifetime imaging. Proc SPIE Photonics West 2006;6091-05.
Hassan M, Riley J, Chernomordik V, Smith P, Pursley R, Lee SB, Capala J, Gandjbakhche AH. Fluorescence lifetime imaging system for in vivo studies. Mol Imaging 2007;6:229-36.
Riley JD, Hassan M, Chernomordik V, Gannot I, Gandjbakhche AH. Time-resolved lifetime fluorescence imaging—an inverse model based on analytical solutions. Proc OSA Biomedical Optics Topical Meeting, Fort Lauderdale, FL, 2006;MH6.
Multimodality imaging techniques to monitor tissue vasculature in Kaposi’s sarcoma lesions
The oncology community is testing a number of novel targeted approaches such as anti-angiogenic, antivascular, and immuno- and gene therapies for use against a variety of cancers. To monitor such therapies, it is desirable to establish techniques to assess tumor vasculature and changes with therapy and to develop and assess noninvasive and quantitative techniques that can both monitor structural changes and assess the functional characteristics or metabolic status of the tumor. For anti-angiogenic therapies, factors associated with blood flow are of particular interest.
We are testing three potential noninvasive imaging techniques to monitor patients undergoing an experimental therapy. The three techniques—infrared thermal imaging (thermography), laser Doppler imaging (LDI), and multispectral imaging—are being tested on subjects with Kaposi’s sarcoma (KS), a highly vascular tumor that occurs frequently among people infected with HIV. Cutaneous KS lesions are easily accessible for such noninvasive techniques and may thus represent a tumor model for assessing certain parameters of angiogenesis. The KS studies are part of ongoing clinical trials under four NCI protocols studying the effects of experimental anti-angiogenic therapies.
Thermography graphically depicts temperature gradients over a given body surface area at a given time and is used to study biological thermoregulatory abnormalities that directly or indirectly influence skin temperature. However, skin temperature is an indirect measure of skin blood flow; the superficial thermal signature of skin is also related to local metabolism. Thus, thermography is best used in conjunction with other techniques. LDI can more directly measure the net blood velocity of small blood vessels in tissue; blood velocity generally increases as blood supply increases during angiogenesis. We recorded thermal patterns with an infrared camera with a uniform sensitivity in the wavelength range of 8 to 12 µm and acquired LDI images by scanning the lesion area of the KS patients at two wavelengths, 690 and 780 nm.
Thermography and LDI have successfully visualized KS lesions; while each technique is capable of measuring an independent parameter, both techniques in combination yielded a strong correlation in a group of 16 patients. Given that LDI measured blood flow distribution in the superficial layer of the skin of the lesion and that the thermal signature provided a combined response of superficial vascularity and metabolic activities of deep tissue, we were able to detect differences among individual lesions. Recently, we added to our clinical study near-infrared spectroscopy (NIRS) in order to assess the pathogenesis of the status and changes of KS lesions during therapy. NIRS is a non-contact and non-invasive method of monitoring changes in blood volume and concentrations of oxygenated and deoxygenated hemoglobin. It can provide early markers for tumor responses and provide information about the pathophysiology of the disease and its changes in response to treatment.
NIRS is most closely related to visual assessment. In collaboration with Stavros Demos, we designed a portable spectral imaging system that captures images with a high-resolution CCD (charge-couple device) camera at six near-infrared wavelengths (700, 750, 800, 850, 900, and 1000 nm). A white light held approximately 15 cm from tissue uniformly illuminated the tissue surface. Using optical filters, we obtained images at the six wavelengths and are now using the intensity images in a mathematical optical model of skin containing two layers: an epidermis and a much thicker, highly scattering dermis. Each layer contains major chromophores that determine absorption in the corresponding layer, and the layers together determine the skin’s total reflectance. We used the effective attenuation of light to determine the effect of the thin epidermis layer on the intensity of the diffusely reflected light, accounting for the epidermis’s absorption coefficient and thickness. We estimated the influence of the much thicker, highly scattering dermis layer on the skin reflectance by a stochastic model of photon migration, e.g., random walk theory, and used multivariate analysis to reconstruct local variations in melanin, oxygenated hemoglobin, and blood volume.
Hassan M, Chernomordik M, Vogel A, Hattery D, Gannot I, Yarchoan R, Gandjbakhche AH. Infrared imaging for tissue characterization and function. In: Bronzino JD, ed. The Biomedical Engineering Handbook, Third Edition, Volume I: Medical Devices and Systems. CRC and IEEE Press, 2006;30-25.
Vogel A, Dasgeb B, Hassan M, Amyot F, Chernomordik V, Tao Y, Demos SG, Wyvill K, Aleman K, Little R, Yarchoan R, Gandjbakhche AH. Using quantitative imaging techniques to assess vascularity in AIDS-related Kaposi’s sarcoma. Conf Proc IEEE Eng Med Biol Soc 2006;232-5.
Vogel A, Hassan M, Amyot F, Chernomordik V, Dasgeb B, Demos SG, Little R, Yarchoan R, TaoY, Gandjbakhche AH. Using multimodality imaging techniques to assess vascularity in AIDS-associated Kaposi’s sarcoma. OSA Biomedical Optics Topical Meeting, Fort Lauderdale, FL, 2006;SG2.
Cellular dynamics of angiogenesis
Tumor-Induced Angiogenesis Model. The process of tumor-induced angiogenesis, in which a growing tumor recruits new vasculature to increase nutrient intake, is crucial to tumor growth. We developed a model of tumor-induced angiogenesis that includes the migratory response of endothelial cells (ECs) to tumor angiogenic factors and the interaction of ECs with the extracellular matrix (ECM). ECs switch among growth, differentiation, motility, or apoptotic behavior in response to the ECM’s local topology and composition. Considering the ECM as a statistically inhomogeneous two-phase random medium, we showed that it can be a natural barrier to angiogenesis. We studied vascular network formation for several ECM distributions and topologies and found a correlation with percolation. A threshold exists under which sprouts cannot reach the tumor, and, during the growth of the vascular network, the attraction exerted by the tumor competes with the preferred path created by the ECM. We also examined the influence of branching on tumor vascularization. Branching is a natural phenomenon that helps the tumor become vascularized. By increasing the number of sprouts (i.e., capillaries), the vascular network increases the probability of reaching the tumor. Our simulations showed that, after two branching events, the vascular network is highly likely to reach the tumor.
Quantitative Assay to Study Cell Trajectory and Morphology in Highly Oriented Collagen Fibers. We prepared thin films of ordered fibrils of collagen I, a major component of the ECM. Collagen films with oriented fibrils mimic the effects of fibroblasts, which naturally tend to orient collagen fibrils in vivo. We prepared the films by modifying a technique previously used to produce collagen I films for studies of cell morphology and intracellular signaling. More specifically, we modified the drying step in order to produce thin monolayers of collagen fibrils with consistent orientations over macroscopic (more than 100 µm) distances. We used Fourier analysis of optical microscopy images to quantify the degree of orientation of the collagen fibrils. We also conducted experiments with vascular ECs and found that cell orientation and migration are well correlated with fibril orientation. Using polarized cells, we showed that an oriented thin collagen film induces natural migration along the fibrils without an attractor. Taken together, our results demonstrate additional functionality and physiological relevance for a class of films that is finding successful application in a variety of cell biology experiments.
We are continuing our studies to characterize the reorganization of the cytoskeleton of different cell lines (endothelial and epithelial) that are in contact with the thin film described above. In addition to classical immunostaining, we labeled microtubules in living cells by incorporating a GFP-a-tubulin fusion protein into the microtubules, which leaves the cells highly motile. Live images of cells showed the reorganization of the cytoskeleton along the fibers during cell locomotion
Directional Guidance by Growth Factor Gradients in Angiogenesis. We study the directional guidance by growth factor gradients in angiogenesis. Large tumors become hypoxic and secrete a growth factor called vascular endothelial growth factor (VEGF). VEGF occurs in several common isoforms, in particularVEGF165 and VEGF189; the most significant difference between these isoforms is that VEGF189 binds to the ECM more rapidly than does VEGF165. The uptake of VEGF by capillaries initiates a sequence of events that leads to the formation of a capillary network. Because the formation of capillary networks is crucial to tumor growth and metastasis, it is of great clinical interest, particularly with respect to understanding which form of VEGF guides chemotactic migration of cells to form a capillary network and identifying the roles played by the ECM and matrix metalloproteases (MMPs). Based on the fact that chemotactic migration is guided by growth factor gradients, we devised a system of reaction-diffusion equations to model the diffusion, binding, and cleavage of VEGF in vivo, with three adjustable parameters: (1) the rate constant for binding to the ECM; (2) the rate of VEGF production, which is known to vary in vivo; and (3) the rate of MMP production by cells proximal to capillaries, which is known to vary in vivo. Our simulations show that rapid binding to the ECM by VEGF189 leads to short-range gradients of matrix-bound VEGF, whereas slower binding by VEGF165 leads to longer-range gradients, consistent with in vivo observations that the vasculature is highly disorganized around tumors producing VEGF189. Only VEGF165 can produce long-range gradients that can guide cell migration to form an efficient and organized network.
We also found that the cleaved form of VEGF, removed from the matrix by the action of MMPs, is distributed with a gradient that points away from the tumor, calling into question whether the cleaved form of VEGF plays a chemotactic role in tumor-induced angiogenesis. This finding is consistent with observations that cleaved VEGF molecules have a weaker chemotactic effect on receptors. Finally, our simulations show that, without MMPs, a gradient of matrix-bound VEGF cannot be sustained. This observation is consistent with findings that the production of MMPs by cells near the parent capillary is necessary to initiate the formation of new vasculature.
Amyot F, Camphausen K, Siavosh A, Sackett D, Gandjbakhche A. Quantitative method to study the network formation of endothelial cells in response to tumor angiogenic factors. IEE Proc Systems Biol 2005;152:61-6.
Amyot F, Small A, Boukari H, Sackett D, Elliott J, McDaniel D, Plant A, Gandjbakhche A. Quantitative assay to study cell motility on collagen film. Biophys J 2007;484A, Suppl. S.
Amyot F, Small A, Boukari H, Sackett D, Elliott J, McDaniel D, Plant A, Gandjbakhche A. Thin films of oriented collagen fibrils for cell motility studies. J Biomed Materials Res Part B: Applied Biomaterials, in press.
Amyot F, Small A, Gandjbakhche A. Stochastic modeling of tumor induced angiogenesis in a heterogeneous medium, the extracellular matrix. Conf Proc IEEE Eng Med Biol Soc 2006;1:3146-9.
Small A, Neagu A, Amyot F, Sackett D, Chernomordik V, Gandjbakhche A. Which form of VEGF can guide endothelial cell migration? Biophys J 2007;647A Suppl. S.
Optimization of multiphoton excitation microscopy
To optimize the signal-to-noise ratio (SNR) for micro-imaging, collaborators at the NHLBI have constructed a device that maximizes the probability of collecting all the scattered and ballistic light generated isotropically at the focal spot of multiphoton-excited emissions (MPEMs). The researchers optically coupled a parabolic reflector (surrounding the sample and top of the objective) to a pair of collimating lenses (above the sample) that redirected the emitted light to a separate detector. The additional optics, combined with the objective, allow the total emission detection (TED) condition to be approached. We performed numeric simulations to study quantitatively the net gain obtained with the new device. Numeric simulations suggest an approximately 10-fold improvement in SNR with TED. Comparisons between the objective detection and TED reveal an enhancement of 8.9 in signal-to-noise ratio (SNR) (77 percent of that predicted) for GFP-labeled brain slices and similar results for fluorescent beads. The increase in SNR can be used with MPEM imaging techniques to improve time resolution, reduce laser power requirements/photodynamic damage, and, in certain cases, enhance detection depth.
TED significantly increases the SNR of MPEM experiments in accord with the numeric simulations. The nearly 10-fold increase in photon collection can have profound implications for MPEM and multiharmonic imaging applications, resulting in numerous enhancements of the MPEM experiment. At constant excitation power, the time required to obtain a given two-dimensional image quality declines by a factor proportional to the increase in photon collection efficiency. In addition, given that TED detectors obey Poisson photon-counting statistical rules, the nine-fold increase in photon collection results in a three-fold increase in SNR. The signal has a squared dependence on excitation power such that the nine-fold enhancement in the SNR with TED can allow a reduction in excitation power by a factor of three without decreasing image quality for the same amount of time. Reports suggest that the relationship between damage and power ranges between quadratic and cubic. Thus, photo damage may be reduced even more than three-fold. It has been suggested that emission collection efficiency limits the depth of detection in biological samples using MPEM. It is difficult to predict precisely the extent to which an improvement in SNR will permit deeper imaging; several factors play a role in the emission and excitation light interaction with the tissue that influence this process (Helmchen and Denk, Nat Methods 2005;2:932; Rothstein et al., Biophys J 2005;88:2165). The excitation efficiency curve may prove to be a tougher limit to overcome as we push farther into dispersive tissue.
TED coupled to MPEM results in a significant improvement in the SNR of the MPEM experiment without degrading image quality. Such a detection system will work best for samples in which emitted light can escape in all directions. Thick or highly absorptive tissues decrease the device’s utility. Nevertheless, for a given fluorophore and a given extent of excitation, TED coupled to MPEM will always surpass the detection limit attained with other optical imaging methods (e.g., confocal microscopy); those methods necessarily collect only a small fraction of the total light emitted.
Combs CA, Smirnov AV, Riley JD, Gandjbakhche AH, Knutson JR, Balaban RS. Optimization of multi-photon excitation microscopy by total emission detection (TED) using a parabolic light reflector. J Microsc 2007;228:330-7.
A simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier
In collaboration with the Center for Devices and Radiological Health at the U.S. Food and Drug Administration, we have designed a novel fiber-optic confocal approach for ultra-high depth resolution (equal to or better than 2 nm) microscopy beyond the diffraction barrier in the subwavelength nanometric range below 200 nm. The approach combines the advanced properties of a simple apertureless single-mode-fiber confocal microscope that provides a highly sensitive diffraction-free Gaussian point light source/receiver and a differential confocal microscope approach that exploits the sharp, diffraction-free slope of the axial confocal response curve.
Within the field of modern bioimaging technologies, high-resolution confocal laser microscopy is an intensively active area of pursuit. The technique provides sharp, high-magnification, three-dimensional imaging with submicron resolution by noninvasive optical sectioning, and rejection of out-of-focus information. However, in the subwavelength nanometric range, which has been the target of considerable effort to obtain quantitative chemical information at cellular/intracellular levels, the newer optical imaging techniques evidence a major limitation related to the fundamental Rayleigh diffraction resolution limit. Various diffraction-free techniques, such as near-field scanning optical microscopy, stimulated emission depletion (STED) fluorescence microscopy, and pinhole-based differential confocal microscopy (DCM), have demonstrated an ability to break the diffraction barrier in optical bioimaging, including confocal microscopy. Our approach to breaking this barrier is based on a fiber-optic confocal microscope compatible with the DCM technique, and with the following specific advanced features. (1) As an apertureless confocal configuration, the fiber-optic design is an effective alternative to conventional pinhole-based confocal systems. It offers several advantages in terms of submicron resolution, high sensitivity to spatial back-reflectance signal displacements, elimination of diffraction/aberration effects, flexibility, miniaturization, and scanning potential. (2) The fiber-optic design involves the specific use of a 2×1 single-mode fiber coupler as a major optical component of the confocal setup. Given its small fiber core dimensions (3–5 µm), the fiber coupler performs several functions simultaneously. It provides effective launching and delivery of the input—continuous wave low-power laser emission—with various transverse mode distributions and wavelengths in the ultraviolet, visible, and infrared ranges. The output—a single-mode fiber tip—serves as a point light source that leads to the formation of a Gaussian laser beam distribution used for precisely collimating and focusing the input laser beam. The same output single-mode fiber tip used as a point light source serves as a point receiver that is highly sensitive to spatial displacements of the focused back-reflectance emission. The fiber coupler provides delivery and intensity sensing of the spatially separated back-reflected optical signals. (3) The design includes a high-numerical-aperture (over 0.8) focusing confocal objective that provides high depth and spatial discriminations, leading to high axial and lateral resolutions. (4) The fiber-optic confocal design is compatible with the pinhole DCM systems based on the use of the sharp diffraction-free slope of the axial confocal response curve rather than the area around the maximum of that curve. We placed the sample slightly away from the focal point area to apply the DCM approach so that its position is at the sharp diffraction-free slope of the axial response curve beyond the diffraction barrier. In this way, the dynamic range and spatial sensitivity increase significantly because the signal light that enters the detecting fiber is highly dependent on the sample position. As a result, we can obtain an ultra-high depth resolution beyond the diffraction limit in the nanometric range. (5) The fiber-optic confocal design employs tools and detecting techniques that possess high signal-to-noise potential such as an intensity-stabilized laser source, a highly sensitive detecting system, and spatial piezoelectric scanning with subnanometric resolution.
The proposed fiber-optic confocal microscopy should provide an ultra-high depth resolution of better than 2 nm. Given the method’s theoretical potential to reach a depth resolution in the 1 nm and subnanometer range, some additional improvements need to be made, such as exploiting lock-in-amplifier detection techniques and close to clean-room environmental conditions.
Ilev I, Waynant R, Gannot I, Gandjbakhche A. A simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier. Rev Sci Instruments 2007;78-093703:1-5.
1 Russian Academy of Sciences
2 University of Maryland, College Park, MD
3 Drexel University, Philadelphia, PA
4 University of Delaware, Newark, DE
5 Ali Shabestari, former Summer Student
6 Sudeh Izadmehr, former Summer Student
COLLABORATORS
Robert Balaban, PhD, Laboratory of Cardiac Energetics, NHLBI, Bethesda, MD
Tiziano Binzoni, PhD, Centre Médical Universitaire, Geneva, Switzerland
Hacène Boukari, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Kevin Camphausen, MD, Radiation Oncology Branch, NCI, Bethesda, MD
Jacek Capala, PhD, Radiation Oncology Branch, NCI, Bethesda, MD
Christian Combs, PhD, Laboratory of Cardiac Energetics, NHLBI, Bethesda, MD
Stavros Demos, PhD, Lawrence Livermore National Laboratory, Livermore, CA
Israel Gannot, PhD, Tel Aviv University, Ramat Aviv, Israel, and The George Washington University, Washington, DC
Jeremy Hebden, PhD, University College London, London, UK
Ilko Ilev, PhD, Office of Science and Engineering Laboratories, FDA, Rockville, MD
Jay Knutson, PhD, Laboratory of Molecular Biophysics, NHLBI, Bethesda, MD
Adrian Neagu, PhD, Victor Babeş University of Medicine and Pharmacy, Timişoara, Romania
Anne Plant, PhD, Chemical Science and Technology Laboratory, NIST, Gaithersburg, MD
Dan Sackett, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Aleksandr Smirnov, PhD, Laboratory of Molecular Biophysics, NHLBI, Bethesda, MD
Paul Smith, PhD, Division of Bioengineering and Physical Science, ORS, NIH, Bethesda, MD
Ronald Waynant, PhD, Office of Science and Engineering Laboratories, FDA, Rockville, MD
George Weiss, PhD, Division of Computational Bioscience, CIT, NIH, Bethesda, MD
Robert Yarchoan, MD, HIV and AIDS Malignancy Branch, NCI, Bethesda, MD
For further information, contact amir@helix.nih.gov.
